DESCRIPTION (taken from the application) The overall goal of our work is to understand the molecular mechanisms through which the development of the pancreatic islet of Langerhans takes place. We have chosen to utilize the zebrafish (Danio rerio) because, unlike other vertebrate model systems, we can directly examine the early embryology of islet development and apply a primary genetic approach to identifying new genes responsible for this process. Zebrafish have the tremendous advantage of being transparent during development, which occurs outside the mother. In addition, mutations can be readily induced and the resulting phenotypes effectively identified, as witnessed by the success of two large scale screens that have recently identified hundreds of mutations effecting vertebrate development. Specifically, by using molecular markers for the endocrine and exocrine pancreas, we will optimize techniques for detection of early islet development. We have isolated the zebrafish insulin gene for use as a probe for whole-mount in situ hybridization. We have identified the insulin mRNA signal in pancreatic anlage present in the early primitive gut as well as in well differentiated islets. We have already demonstrated that whole-mount in situ hybridization with the insulin probe can identify genetic abnormalities of islet development proving its facility as a specific screening tool in genetic screens. For example, in the notocord mutant floating head we have shown that insulin expression and anatomical development of the pancreas is severely altered. This strategy will be subsequently applied to our primary aim; our participation in a large scale, multi-investigator zebrafish screening project using retroviral mutagenesis. Due to the relative ease in isolating responsible genes provided by this approach, a real opportunity exists to isolate new genes responsible for beta cell differentiation. The pattern of expression and the functional role of any newly identified genes will first be extensively studied in zebrafish embryos and adults. Genes whose mutation causes distinct effects on zebrafish islet development will then be examined in the context of the mammal in collaboration with investigators expert in the embryology of the mouse pancreas by determining the phenotypes caused by targeted inactivation of the homologous mouse genes. We also propose to generate transgenic zebrafish lines that express green fluorescent protein in mature and developing pancreatic beta cells to serve as reporter organisms to facilitate genetic screening and precursor cell identification. We predict that insights into the control of islet development and growth will in this way be translated into practical approaches for identifying early, multipotential islet stem cells, as well as generating strategies to create and expand human islet tissue in vitro for therapeutic transplantation into type I diabetic patients.